Partial deletion of human alpha-galactosidase A gene in Fabry disease: direct repeat sequences as a possible cause of slipped mispairing

A partial deletion involving exon 3 associated with a single base change (A to C) was found in the alpha-galactosidase A gene of a hemizygous male Fabry patient and his mother, a heterozygous proband. This 402-bp deletion was flanked by 6-bp direct repeat sequences, and the intervening portion was found to have unique complementary sequences. These specific structures may have promoted "slipped mispairing" in this family.     

A novel single nucleotide polymorphism altering stability and activity of CYP2a6

CYP2A6 is known as a major cytochrome P450 (CYP) responsible for the oxidation of nicotine and coumarin in humans. In this study, we explored genetic polymorphisms, which reduce CYP2A6 activity in Japanese. Two novel mutations in exon 9 of the CYP2A6 gene were found. A single nucleotide polymorphism of T1412C and G1454T resulted in Ile471Thr and Arg485Leu substitution, respectively. The frequency of the former variant allele was considerably high (15.7%), while the latter variant appeared to be a rare polymorphism. Heterologous expression of CYP2A6 using a cDNA possessing C instead of T-base at codon 471 in Escherichia coli caused remarkable reduction of the stability of holoenzyme at 37 degrees C. Furthermore, this variant enzyme almost lacked nicotine C-oxidase activity, although coumarin 7-hydroxylase activity was still observed. These data suggest that individuals homozygous for the T1412C variant allele or heterozygous for this and a defect allele such as the CYP2A6*4 may be poor metabolizer of nicotine, but not coumarin.     

Average values of a dissimilarity measure not requiring sequence alignment are twice the averages of conventional mismatch counts requiring sequence alignment for a computer-generated model system

Summary Three measures of sequence dissimilarity have been compared on a computer-generated model system in which substitutions in random sequences were made at randomly selected sites and the replacement character was chosen at random from the set of characters different from the original occupant of the site. The three measures were the conventionalmmismatch count between aligned sequences (AMC=m) and two measures not requiring prior sequence alignment. The latter two measures were the squared Euclidean distance between vectors of counts of t-tuples (t=1–6) of characters in the two sequences (multiplet distribution distances or MDD=d) and counts of characters not covered by word structures of statistically significant length common to the two sequences (common long words or CLW=SIB, SIS, or SAB). Average MDD distances were found to be two times average mismatch counts in the simulated sequences for all values of t from 1 to 6 and all degrees of substitution from one per sequence to so many as to produce, effectively, random sequences. This simple relation held independently of sequence length and of sequence composition. The relation was confirmed by exact results on small model systems and by formal asymptotic results in the limit of so few substitutions that no double hits occur and in the limit of two random sequences. The coefficient of variation for MDD distances was greater than that for mismatch counts for singlets but both measures approached the same low value for sextets. Needleman-Wunsch alignment produced incorrect mismatch counts at higher degrees of substitution. The model satisfied the conditions for the derivation of the Jukes-Cantor asymptotic adjustment, but its application produced increasingly bad results with increasing degrees of substitution in accord with earlier results on model and natural sequences. This fact was a consequence of the increase with increasing degrees of substitution of the sensitivity of the adjustment to error in the observations. Average CLW distances for a variety of common word structures were more or less parallel to MDD distances for appropriately long t-tuples. These results on model systems supported the validity of the two dissimilarity measures not requiring sequence alignment that was found in earlier work on natural sequences (Blaisdell 1989).     

水牛、大额牛和牦牛抑制素α亚基编码基因外显子1多态性分析

为了揭示牛科物种INHA基因的遗传特征,该文采用PCR产物直接测序法对水牛、大额牛和牦牛INHA基因外显子1及其侧翼序列进行多态性检测,并结合已发表的包括牛科物种在内的一些哺乳动物数据进行了比较分析。结果表明,在水牛INHA基因外显子1中存在c.73C>A替换,为同义替换,河流型和沼泽型水牛编码产物一致;在大额牛的INHA基因外显子1中发现c.62C>T、c.187G>A替换,分别引起INHA中氨基酸发生p.P21L、p.V63M改变,两者均为相同性质氨基酸的替换;在牦牛中发现c.62C>T、c.129A>G替换,前者也引起编码氨基酸发生p.P21L替换,后者为同义替换。在INHA基因5’侧翼区所测出的序列中,水牛、大额牛和牦牛等物种内均未发现SNP位点,但在种间发现存在c.-6T>G的替换,大额牛、牦牛和普通牛均为c.-6G,而水牛为c.-6T。在INHA基因内含子中,水牛的第31～36位核苷酸处发现有6个碱基的缺失,即c.262+31₂62+36delTCTGAC;该位点在河流型水牛中野生型（+/+）占主体,而在沼泽型水牛中则缺失型（-/-）占主体。在大额牛、牦牛和普通牛等其它牛科物种的内含子中均未发现该缺失,但与水牛相比,大额牛、牦牛和普通牛内含子中发现缺失c.262+78₂62+79delTG。序列比对显示,INHA基因外显子1序列中c.43A和c.67G为水牛中所特有,而c.173A和c.255G为大额牛、牦牛和普通牛所共有,c.24C、c.47G、c.174T和c.206T为山羊所特有。大额牛、牦牛和普通牛间INHA基因外显子1序列差异较小,而山羊和水牛与它们间的差异相对较大。     

HPRTSardinia: a new point mutation causing HPRT deficiency without Lesch-Nyhan disease

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency always causing hyperuricemia presents various degrees of neurological manifestations, the most severe which is Lesch-Nyhan syndrome. The HPRT gene is situated in the region Xq26-q27.2 and consists of 9 exons. At least 300 different mutations at different sites in the HPRT coding region from exon 1 to exon 9 have been identified. A new mutation in the HPRT gene has been determined in one patient with complete deficiency of erythrocyte activity, with hyperuricemia and gout but without Lesch-Nyhan disease. Analysis of cultured fibroblasts revealed minimal residual HPRT activity mainly when guanine was the substrate. Genomic DNA sequencing demonstrated patient's mother heterozygosity for the mutation and no mutation in her brother. The mutation consists in a C-->T transversion at cDNA base 463 (C463T) in exon 6, resulting in proline to serine substitution at codon 155 (P155S). This mutation had not been reported previously and has been designated HPRT(Sardinia). The mutation identified in this patient allows some expression of functional enzyme in nucleated cells such as fibroblasts, indicating that such cell type may add further information to conventional blood analysis. A multicentre survey gathering patients with variant neurological forms could contribute to understand the pathophysiology of the neurobehavioral symptoms of HPRT deficiency.     

The molecular basis of homocystinuria due to cystathionine beta-synthase deficiency in Italian families, and report of four novel mutations.

Four new mutations in the cystathionine beta-synthase (CBS) gene have been identified in Italian patients with homocystinuria. The first mutation is a G-to-A transition at base 374 in exon 3, causing an arginine-to-glutamic acid substitution at position 125 of the protein (R125Q). This mutation has been found in homozygosity in a patient partially responsive to pyridoxine treatment. The second mutation is a C-to-T transition at base 770 in exon 7, causing a threonine-to-methionine substitution at amino acid 257 of the protein (T257M). This mutation has been observed in homozygosity in a patient nonresponsive to the cofactor treatment. The third mutation, found in heterozygosity in a patient responsive to pyridoxine treatment, is an insertion of 68 bp in exon 8 at base 844, which introduces a premature termination codon. The fourth mutation is C-to-T transition in exon 2 at base 262, causing a proline-to-serine substitution at position 88 of the protein (P88S). This mutation is carried on a single allele in three affected sisters responsive to the cofactor treatment. In addition, six previously reported mutations (A114V, E131D, P145L, I278T, G307S, and A1224-2C) have been tested in 14 independent Italian families. Mutations A114V and I278T are carried by three and by seven independent alleles, respectively. The other four mutations--including G307S and A1224-2C, common among northern European patients--have not been detected.     

Human adenylate kinase deficiency associated with hemolytic anemia. A single base substitution affecting solubility and catalytic activity of the cytosolic adenylate kinase

Adenylate kinase deficiency in the erythrocyte is a rare genetic disorder associated with hemolytic anemia. To determine the molecular basis of this disorder, we first cloned the normal gene encoding human cytosolic adenylate kinase (AK1) and determined the structure. The gene was 12 kilobase pairs long and was split into 7 exons. The structures of 5'- and 3'-flanking regions were determined by primer extension and RNA blot analysis. The results showed that two species of mRNA with 0.9 and 2.5 kilobases, which differed at the 3'-end portion, were generated by the AK1 gene. Alu sequences were found in the largest intron (intron 5) and in the noncoding region of exon 7. Next, both alleles of the AK1 gene were cloned from DNA of a patient bearing the adenylate kinase deficiency and their nucleotide sequences determined. A transition (C----T) was found in exon 6 on an allele, which resulted in an Arg to Trp (CGG----TGG) substitution at the 128th residue of AK1. Since chicken AK1 is highly homologous to human AK1 with respect to the amino acid sequence, we introduced an Arg to Trp substitution to chicken AK1 at the same position by oligodeoxynucleotide-directed mutagenesis. The mutant chicken AK1 expressed in Escherichia coli showed a reduced catalytic activity as well as a decreased solubility and a change in affinity to phosphocellulose. Thus it was considered that the observed C----T transition was a cause of the decreased AK1 activity of the patient's erythrocyte. Analysis on phosphocellulose chromatography of erythrocyte AK1 of the patient and parents revealed that the patient's mutant allele was derived from the mother.     

New 6-butylamino-6-deoxycellulose and 6-deoxy-6-pyridiniumcellulose derivatives with highest regioselectivity and completeness of reaction

This paper investigates the nucleophilic substitution (S(N)) reactions of tosylcellulose with butylamine and pyridine, respectively. The S(N) reactions of tosylcellulose 1 (DS(Total) 2.02; DS(C-6) 1.0) with butylamine carried out at 25, 50, 75 and 100 degrees C in both dimethyl sulfoxide (DMSO) and pure butylamine showed that the regioselectivity of substitution at C-6 of cellulose is temperature dependent: the highest regioselectivity at C-6 can be reached at 25 and 50 degrees C; substitution at C-2 also occurred at 75 and 100 degrees C. The substitution speed in pure butylamine is greater than that in the presence of DMSO. A complete and regioselective substitution at C-6 with a DS of 1.0 was obtained under the conditions of 50 degrees C, 40 h in butylamine. The substitution reactions of 1 with pyridine carried out at 25, 50, 75 and 100 degrees C for 24h in DMSO did not occur. In contrast to this the S(N) reactions done in pure pyridine showed that a temperature- and steric-dependent, regioselective substitution took place at C-6 at temperatures from 25 to 145 degrees C. The highest regioselectivity and completeness at C-6 can be obtained at 100 degrees C for 90 h, whereas at 145 degrees C substitution also occurs at C-2. The results were proved by 1H NMR and 13C NMR spectroscopy.     

DNA sequence abnormalities of human glucose-6-phosphate dehydrogenase variants

Over 400 supposedly biochemically and genetically distinct variants of glucose-6-phosphate dehydrogenase (G6PD) have been described in the past. In order to investigate these variants at the DNA sequence level we have now determined the relevant sequences of introns of G6PD and describe a method which allows us to rapidly determine the sequence of the entire coding region of G6PD. This technique was applied to six variants that cause G6PD deficiency to be functionally so severe as to result in nonspherocytic hemolytic anemia. Although the patients were all unrelated, G6PD Marion, Gastonia, and Minnesota each had identical mutations, a G----T at nucleotide (nt) 637 in exon 6 leading to a Val----Leu substitution at amino acid 213. The mutations of Nashville and Anaheim were identical to each other, viz. G----A at nt 1178 in exon 10 producing a Arg----His substitution at amino acid 393. G6PD Loma Linda had a C----A substitution at nt 1089 in exon 10, producing a Asn----Lys change at amino acid 363. The results confirm our earlier results suggesting that the NADP-binding site is in a small region of exon 10 and suggest the possibility that this area is also concerned with the binding of glucose-6-P.     

A novel exon mutation in the human beta-hexosaminidase beta subunit gene affects 3' splice site selection.

The molecular basis of a dramatically decreased steady state level of beta-hexosaminidase beta subunit mRNA in a patient with juvenile Sandhoff disease was investigated. Nucleotide sequence analysis of the HEXB gene coding for the beta subunit revealed two single base substitutions, one in exon 2 (A to G, a known polymorphism) and the other in exon 11 (C to T). Analysis of the beta subunit mRNA species demonstrated activation of a cryptic splice site in exon 11 as well as skipping of the exon. A transfection assay using a chimeric gene containing intron 10 flanked by cDNA sequences carrying the mutation confirmed that the single base substitution located at position 8 of exon 11 inhibits the selection of the normal 3' splice site. The results demonstrate a new type of exon mutation affecting 3' splice site selection.     

Exonic splicing enhancers contribute to the use of both 3' and 5' splice site usage of rat beta-tropomyosin pre-mRNA

The rat beta-tropomyosin gene encodes two tissue-specific isoforms that contain the internal, mutually exclusive exons 6 (nonmuscle/smooth muscle) and 7 (skeletal muscle). We previously demonstrated that the 3' splice site of exon 6 can be activated by introducing a 9-nt polyuridine tract at its 3' splice site, or by strengthening the 5' splice site to a U1 consensus binding site, or by joining exon 6 to the downstream common exon 8. Examination of sequences within exons 6 and 8 revealed the presence of two purine-rich motifs in exon 6 and three purine-rich motifs in exon 8 that could potentially represent exonic splicing enhancers (ESEs). In this report we carried out substitution mutagenesis of these elements and show that some of them play a critical role in the splice site usage of exon 6 in vitro and in vivo. Using UV crosslinking, we have identified SF2/ASF as one of the cellular factors that binds to these motifs. Furthermore, we show that substrates that have mutated ESEs are blocked prior to A-complex formation, supporting a role for SF2/ASF binding to the ESEs during the commitment step in splicing. Using pre-mRNA substrates containing exons 5 through 8, we show that the ESEs within exon 6 also play a role in cooperation between the 3' and 5' splice sites flanking this exon. The splicing of exon 6 to 8 (i.e., 5' splice site usage of exon 6) was enhanced with pre-mRNAs containing either the polyuridine tract in the 3' splice site or consensus sequence in the 5' splice site around exon 6. We show that the ESEs in exon 6 are required for this effect. However, the ESEs are not required when both the polyuridine and consensus splice site sequences around exon 6 were present in the same pre-mRNA. These results support and extend the exon-definition hypothesis and demonstrate that sequences at the 3' splice site can facilitate use of a downstream 5' splice site. In addition, the data support the hypothesis that ESEs can compensate for weak splice sites, such as those found in alternatively spliced exons, thereby providing a target for regulation.     

Identification of a single base change in a new human mutant glucose-6-phosphate dehydrogenase gene by polymerase-chain-reaction amplification of the entire coding region from genomic DNA.

We report the characterization at the molecular level of a mutant glucose-6-phosphate dehydrogenase (G6PD) gene in a Greek boy who presented with a chronic non-spherocytic haemolytic anaemia. In order to identify the mutation from a small amount of patient material, we adopted an approach which by-passes the need to construct a library by using the polymerase chain reaction. The entire coding region was amplified in eight sections, with genomic DNA as template. The DNA fragments were then cloned in an M13 vector and sequenced. The only difference from the sequence of normal G6PD was a T----G substitution at nucleotide position 648 in exon 7, which predicts a substitution of leucine for phenylalanine at amino acid position 216. This mutation creates a new recognition site for the restriction nuclease BalI. We confirmed the presence of the mutation in the DNA of the patient's mother, who was found to be heterozygous for the new BalI site. This is the first transversion among the point mutations thus far reported in the human G6PD gene.     

A missense mutation found in human lactate dehydrogenase-B (H) variant gene

A human lactate dehydrogenase-B mutant gene was isolated from a genomic DNA library constructed from a patient with unstable LDH-B (heart) subunit. The nucleotide sequences of seven protein-coding exons were determined and a single nucleotide substitution of G by A at Arg codon CGC in exon 4 was found. This mutation results in an amino-acid replacement of a conserved arginine by histidine at the residue "173," which is involved in an anion-binding site at the P-axis of LDH subunits.     

HPRTSardinia: a new point mutation causing HPRT deficiency without Lesch–Nyhan disease

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency always causing hyperuricemia presents various degrees of neurological manifestations, the most severe which is Lesch–Nyhan syndrome. The HPRT gene is situated in the region Xq26-q27.2 and consists of 9 exons. At least 300 different mutations at different sites in the HPRT coding region from exon 1 to exon 9 have been identified. A new mutation in the HPRT gene has been determined in one patient with complete deficiency of erythrocyte activity, with hyperuricemia and gout but without Lesch–Nyhan disease. Analysis of cultured fibroblasts revealed minimal residual HPRT activity mainly when guanine was the substrate. Genomic DNA sequencing demonstrated patient's mother heterozygosity for the mutation and no mutation in her brother. The mutation consists in a C→T transversion at cDNA base 463 (C463T) in exon 6, resulting in proline to serine substitution at codon 155 (P155S). This mutation had not been reported previously and has been designated HPRT_Sardinia. The mutation identified in this patient allows some expression of functional enzyme in nucleated cells such as fibroblasts, indicating that such cell type may add further information to conventional blood analysis. A multicentre survey gathering patients with variant neurological forms could contribute to understand the pathophysiology of the neurobehavioral symptoms of HPRT deficiency.     

Allotypes of the constant region of the rabbit T cell receptor beta-2 chain

Our laboratory previously reported that there was restriction fragment length polymorphism of TCR C beta genes in rabbits. EcoRI digests of DNA from different rabbits gave fragments of 9 and 6 kb (C beta a) or 14 and 6 kb (C beta b) that hybridized to a C beta cDNA probe. We also reported that the 9- and 14-kb types segregated as Mendelian traits and that there were allotypic differences in the first exon of the C beta 1 genes of C beta a and C beta b animals. Here we report the DNA sequence of the C beta 2 gene present in the 6-kb EcoRI fragment from a C beta b animal and compare the exon sequences with that of a cDNA from a C beta a animal. We find replacement changes in the first and third exons that probably represent allotypic forms of the rabbit C beta 2 gene. The genomic DNA 5' of exon 1 of both beta 1 and beta 2 contain alternating purine/pyrimidine repeat sequences. The genomic C beta 2 has an open reading frame of 69 amino acids in frame with exon 1 similar to a longer one previously found 5' of exon 1 of C beta 1. Further 5' of this region, rabbit C beta 1 and C beta 2 DNA sequences are only about 66% similar. Both the C beta 1 and C beta 2 sequences have two chi sequences; one in exon 1 with a perfect match and one in the intron downstream of exon 1 with one mismatch. Alternating purine/pyrimidine repeats and chi sequences found in rabbit C beta 1 and C beta 2 genes may have contributed to process(es) of gene duplication and/or conversion.     

Two commonly occurring nucleotide base substitutions in Chinese G6PD variants

Using a direct PCR sequencing technique, we have identified two DNA base substitutions in 8 different biochemical G6PD variants of Chinese origin. Neither one of these abnormalities has been reported in other ethnic groups. An abnormality (C1) of G to T substitution at cDNA 1376 causing an amino acid change from Arg to Leu has been found in 3 variants. Another abnormality (C2) of G to A substitution at cDNA 1388 causing an amino acid change from Arg to His has been found in 5 variants. Both C1 and C2 are located in exon 12 of the G6PD gene and are only 12 base pairs apart. However, C1 is associated with a significant increase in the deamino-NADP utilization rate, whereas C2 is not. Taken together, our data suggest that C1 and C2 are very common among Chinese with a G6PD deficiency and exon 12 may define an important functional domain of the human G6PD.     

G to T transversion at position +5 of a splice donor site causes skipping of the preceding exon in the type III procollagen transcripts of a patient with Ehlers-Danlos syndrome type IV.

We identified a splicing mutation in a patient with Ehlers-Danlos syndrome type IV, a heritable connective tissue disorder associated with dysfunctions of type III collagen. The mutation was first localized in the patient's type III procollagen mRNA by amplifying the reverse transcribed product in several overlapping fragments using the polymerase chain reaction. Amplified products spanning exon 24-26 sequences displayed two distinct fragments, one of normal size and the other lacking the 99 base pairs of exon 25. Sequencing of amplified genomic products identified a G to T transversion at position +5 of the splice donor site of intron 25 in one of the patient's procollagen III genes. Expression of allelic minigene constructs correlated the T for G substitution with skipping of exon 25 sequences. Like previously characterized splicing mutations in other collagen genes, lowering the temperature at which the patient's fibroblasts were incubated nearly abolished exon skipping. As a part of this study, we also identified a highly polymorphic, intronic DNA sequence whose different allelic forms can be detected easily by the polymerase chain reaction technique.